Quantitative Analysis of Isopimpinellin from Ammi majus L. Fruits and Evaluation of Its Biological Effect on Selected Human Tumor Cells.

Ammi majus L. (Apiaceae) is a medicinal plant with a well-documented history in phytotherapy. The aim of the present work was to isolate isopimpinellin (5,8-methoxypsoralen; IsoP) from the fruit of this plant and evaluate its biological activity against selected tumor cell lines. The methanol extract obtained with the use of an accelerated solvent extraction (ASE) method was the most suitable for the quantitative analysis of coumarins in the A. majus fruit matrix. The coumarin content was estimated by RP-HPLC/DAD, and the amount of IsoP was found to be 404.14 mg/100 g dry wt., constituting 24.56% of the total coumarin fraction (1.65 g/100 g). This, along with the presence of xanthotoxin (368.04 mg/100 g, 22.36%) and bergapten (253.05 mg/100 g, 15.38%), confirmed A. majus fruits as an excellent source of these compounds. IsoP was isolated (99.8% purity) by combined liquid chromatography/centrifugal partition chromatography (LC/CPC) and tested for the first time on its antiproliferative activity against human colorectal adenocarcinoma (HT29, SW620), osteosarcoma (Saos-2, HOS), and multiple myeloma (RPMI8226, U266) cell lines. MTT assay results (96 h incubation) demonstrated a dose- and cell line-dependent decrease in cell proliferation/viability, with the strongest effect of IsoP against the Saos-2 cell line (IC50; 42.59 µM), medium effect against U266, HT-29, and RPMI8226 (IC50 = 84.14, 95.53, and 105.0 µM, respectively), and very weak activity against invasive HOS (IC50; 321.6 µM) and SW620 (IC50; 711.30 µM) cells, as well as normal human skin fibroblasts (HSFs), with IC50; 410.7 µM. The mechanistic study on the Saos-2 cell line showed that IsoP was able to reduce DNA synthesis and trigger apoptosis via caspase-3 activation. In general, IsoP was found to have more potency towards cancerous cells (except for HOS and SW620) than against healthy cells. The Selective Index (SI) was determined, underlining the higher selectivity of IsoP towards cancer cells compared to healthy cells (SI = 9.62 against Saos-2). All these results suggest that IsoP might be a promising molecule in the chemo-prevention and treatment of primary osteosarcoma.

Thalidomide derivatives as nanomolar human neutrophil elastase inhibitors: Rational design, synthesis, antiproliferative activity and mechanism of action.

Here, we rationally designed a human neutrophil elastase (HNE) inhibitors 4a-4f derived from thalidomide. The HNE inhibition assay showed that synthesized compounds 4a, 4b, 4e and 4f demonstrated strong HNE inhibiton properties with IC50 values of 21.78-42.30 nM. Compounds 4a, 4c, 4d and 4f showed a competitive mode of action. The most potent compound 4f shows almost the same HNE inhibition as sivelestat. The molecular docking analysis revealed that the strongest interactions occur between the azetidine-2,4-dione group and the following three aminoacids: Ser195, Arg217 and His57. A high correlation between the binding energies and the experimentally determined IC50 values was also demonstrated. The study of antiproliferative activity against human T47D (breast carcinoma), RPMI 8226 (multiple myeloma), and A549 (non-small-cell lung carcinoma) revealed that designed compounds were more active compared to thalidomide, pomalidomide and lenalidomide used as the standard drugs. Additionally, the most active compound 4f derived from lenalidomide induces cell cycle arrest at the G2/M phase and apoptosis in T47D cells.

Evaluation of the Biological Effect of Non-UV-Activated Bergapten on Selected Human Tumor Cells and the Insight into the Molecular Mechanism of Its Action.

There is some evidence that non-photoactivated psoralens may be active against breast and colon tumor cells. Therefore, we evaluated the antiproliferative, proapoptotic, and anti-migrative effect of 5-methoxypsoralen (5-MOP) isolated from Peucedanum tauricum MB fruits in human colorectal adenocarcinoma (HT-29 and SW620), osteosarcoma (Saos-2 and HOS), and multiple myeloma (RPMI8226 and U266). Dose- and cell-line-dependent effects of 5-MOP on viability and proliferation were observed, with the strongest inhibitory effect against Saos-2 and a moderate effect against the HOS, HT-29, and SW620 cells. Multiple myeloma showed low sensitivity. The high viability of human normal cell cultures (HSF and hFOB) in a wide range of 5-MOP concentrations tested (6.25-100 µM) was confirmed. Moreover, the migration of treated Saos-2, SW620, and HT-29 cell lines was impaired, as indicated via a wound healing assay. Flow cytometry analysis conducted on Saos-2 cells revealed the ability of 5-MOP to block the cell cycle in the G2 phase and trigger apoptosis, which was accompanied by a loss of mitochondrial membrane potential, caspases (-9 and -3) activation, the altered expression of the Bax and Bcl-2 proteins, and decreased AKT phosphorylation. This is the first report evaluating the antiproliferative and antimigratory impact of non-UV-activated bergapten on the abovementioned (except for HT-29) tumor cells, which provides new data on the potential role of 5-MOP in inhibiting the growth of various types of therapeutic-resistant cancers.

Synergistic Antifungal Interactions between Antibiotic Amphotericin B and Selected 1,3,4-thiadiazole Derivatives, Determined by Microbiological, Cytochemical, and Molecular Spectroscopic Studies.

In recent years, drug-resistant and multidrug-resistant fungal strains have been more frequently isolated in clinical practice. This phenomenon is responsible for difficulties in the treatment of infections. Therefore, the development of new antifungal drugs is an extremely important challenge. Combinations of selected 1,3,4-thiadiazole derivatives with amphotericin B showing strong synergic antifungal interactions are promising candidates for such formulas. In the study, microbiological, cytochemical, and molecular spectroscopy methods were used to investigate the antifungal synergy mechanisms associated with the aforementioned combinations. The present results indicate that two derivatives, i.e., C1 and NTBD, demonstrate strong synergistic interactions with AmB against some Candida species. The ATR-FTIR analysis showed that yeasts treated with the C1 + AmB and NTBD + AmB compositions, compared with those treated with single compounds, exhibited more pronounced abnormalities in the biomolecular content, suggesting that the main mechanism of the synergistic antifungal activity of the compounds is related to a disturbance in cell wall integrity. The analysis of the electron absorption and fluorescence spectra revealed that the biophysical mechanism underlying the observed synergy is associated with disaggregation of AmB molecules induced by the 1,3,4-thiadiazole derivatives. Such observations suggest the possibility of the successful application of thiadiazole derivatives combined with AmB in the therapy of fungal infections.

Heterogeneous Cellular Response of Primary and Metastatic Human Gastric Adenocarcinoma Cell Lines to Magnoflorine and Its Additive Interaction with Docetaxel.

Gastric cancer is the most common cancer and remains the leading cause of cancer death worldwide. In this study, the anticancer action of magnoflorine isolated via counter-current chromatography from the methanolic extract of Berberis vulgaris root against gastric cancer in models of primary ACC-201 and AGS and metastatic MKN-74 and NCI-N87 cell lines was analyzed. Cell viability and proliferation were tested through the use of MTT and BrdU tests, respectively. Cell cycle progression and apoptosis were evaluated using flow cytometry. The interaction of magnoflorine and docetaxel has been examined through isobolographic analysis. Moreover, potential toxicity was verified in zebrafish in an in vivo model. Gastric cancer cell lines revealed different responses to magnoflorine treatment with regard to viability/proliferation, apoptosis induction and cell cycle inhibition without any undesirable changes in the development of larval zebrafish at the tested concentrations. What is more, magnoflorine in combination with docetaxel produced an additive pharmacological interaction in all studied gastric cancer cell lines, which may suggest a complementary mechanism of action of both compounds. Taken together, these findings provide a foundation for the possibility of magnoflorine as a potential therapeutic approach for gastric cancer and merits further investigation, which may pave the way for clinical uses of magnoflorine.

Emodin-8-O-Glucoside-Isolation and the Screening of the Anticancer Potential against the Nervous System Tumors.

Emodin-8-O-glucoside (E-8-O-G) is a glycosylated derivative of emodin that exhibits numerous biological activities, including immunomodulatory, anti-inflammatory, antioxidant, hepatoprotective, or anticancer activities. However, there are no reports on the activity of E-8-O-G against cancers of the nervous system. Therefore, the aim of the study was to investigate the antiproliferative and cytotoxic effect of E-8-O-G in the SK-N-AS neuroblastoma, T98G human glioblastoma, and C6 mouse glioblastoma cancer cells. As a source of E-8-O-G the methanolic extract from the aerial parts of Reynoutria japonica Houtt. (Polygonaceae) was used. Thanks to the application of centrifugal partition chromatography (CPC) operated in the descending mode using a mixture of petroleum ether:ethyl acetate:methanol:water (4:5:4:5 v/v/v/v) and a subsequent purification with preparative HPLC, E-8-O-G was obtained in high purity in a sufficient quantity for the bioactivity tests. Assessment of the cancer cell viability and proliferation were performed with the MTT (3-(bromide 4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium), CTG (CellTiter-Glo®) and BrdU (5-bromo-2'-deoxyuridine) assays, respectively. E-8-O-G inhibits the viability and proliferation of SK-N-AS neuroblastoma, T98G human glioblastoma multiforme, and C6 mouse glioblastoma cells dose-dependently. E-8-O-G seems to be a promising natural antitumor compound in the therapy of nervous system tumors.

New Borane-Protected Derivatives of α-Aminophosphonous Acid as Anti-Osteosarcoma Agents: ADME Analysis and Molecular Modeling, In Vitro Studies on Anti-Cancer Activities, and NEP Inhibition as a Possible Mechanism of Anti-Proliferative Activity.

Many organophosphorus compounds (OPs), especially various α-aminophosphonates, exhibit anti-cancer activities. They act, among others, as inhibitors of the proteases implicated in cancerogenesis. Thesetypes of inhibitors weredescribed, e.g., for neutral endopeptidase (NEP) expressed in different cancer cells, including osteosarcoma (OS). The aim of the present study isto evaluate new borane-protected derivatives of phosphonous acid (compounds 1-7) in terms of their drug-likeness properties, anti-osteosarcoma activities in vitro (against HOS and Saos-2 cells), and use as potential NEP inhibitors. The results revealed that all tested compounds exhibited the physicochemical and ADME properties typical for small-molecule drugs. However, compound 4 did not show capability of blood-brain barrier penetration (Lipinski and Veber rules;SwissAdme tool). Moreover, the α-aminophosphonite-boranes (compounds 4-7) exhibited stronger anti-proliferative activity against OS cells than the other phosphonous acid-borane derivatives (compounds 1-3),especially regarding HOS cells (MTT assay). The most promising compounds 4 and 6 induced apoptosis through the activation of caspase 3 and/or cell cycle arrest at the G2 phase (flow cytometry). Compound 4 inhibited the migration and invasiveness of highly aggressive HOS cells (wound/transwell and BME-coated transwell assays, respectively). Additionally, compound 4 and, to a lesser extent, compound 6 inhibited NEP activity (fluorometric assay). This activity of compound 4 was involved in its anti-proliferative potential (BrdU assay). The present study shows that compound 4 can be considered a potential anti-osteosarcoma agent and a scaffold for the development of new NEP inhibitors.

Effect of Antibiotic Amphotericin B Combinations with Selected 1,3,4-Thiadiazole Derivatives on RPTECs in an In Vitro Model.

4-(5-methyl-1,3,4-thiadiazole-2-yl) benzene-1,3-diol (C1) and 4-[5-(naphthalen-1-ylmethyl)-1,3,4-thiadiazol-2-yl] benzene1,3-diol (NTBD) are representative derivatives of the thiadiazole group, with a high antimycotic potential and minimal toxicity against normal human fibroblast cells. The present study has proved its ability to synergize with the antifungal activity of AmB. The aim of this work was to evaluate the cytotoxic effects of C1 or NTBD, alone or in combination with AmB, on human renal proximal tubule epithelial cells (RPTECs) in vitro. Cell viability was assessed with the MTT assay. Flow cytometry and spectrofluorimetric techniques were used to assess the type of cell death and production of reactive oxygen species (ROS), respectively. The ELISA assay was performed to measure the caspase-2, -3, and -9 activity. ATR-FTIR spectroscopy was used to evaluate biomolecular changes in RPTECs induced by the tested formulas. The combinations of C1/NTBD and AmB did not exert a strong inhibitory effect on the viability/growth of kidney cells, as evidenced by the negligible changes in the apoptotic/necrotic rate and caspase activity, compared to the control cells. Both NTBD and C1 displayed stronger anti-oxidant activity when combined with AmB. The relatively low nephrotoxicity of the thiadiazole derivative combinations and the protective activity against AmB-induced oxidative stress may indicate their potential use in the therapy of fungal infections.

Alpha Ketoglutarate Downregulates the Neutral Endopeptidase and Enhances the Growth Inhibitory Activity of Thiorphan in Highly Aggressive Osteosarcoma Cells.

Since natural substances are widely explored as epigenetic modulators of gene expression and epigenetic abnormalities are important causes of cancerogenesis, factors with pro-tumor activities subjected to epigenetic control, e.g., neutral endopeptidase (NEP, neprilysin), are promising anticancer targets for potential therapies acting via epigenetic regulation of gene expression. Alpha-ketoglutarate (AKG) is a naturally occurring co-substrate for enzymes involved in histone and DNA demethylation with suggested anti-cancer activity. Hence, we investigated a potential effect of AKG on the NEP expression in cells derived from various cancers (cervical, colon, osteosarcoma) and normal epithelial cells and osteoblasts. Moreover, the overall methylation status of histone H3 was explored to establish the molecular target of AKG activity. Additionally, it was investigated whether AKG in combination with thiorphan (NEP specific inhibitor) exhibited enhanced anticancer activity. The results revealed that AKG downregulated the expression of NEP at the protein level only in highly aggressive osteosarcoma HOS cells (flow cytometry and fluorometric assays), and this protease was found to be involved in AKG-induced growth inhibition in osteosarcoma cells (siRNA NEP silencing, BrdU assay, flow cytometry). Unexpectedly, AKG-induced hypermethylation of H3K27 in HOS cells, which was partially dependent on EZH2 activity. However, this effect was not implicated in the AKG-induced NEP downregulation (flow cytometry). Finally, the combined treatment with AKG and thiorphan was shown to significantly enhance the growth inhibitory potential of each one towards HOS cells (BrdU assay). These preliminary studies have shown for the first time that the downregulation of NEP expression is a promising target in therapies of NEP-implicating HOS cells. Moreover, this therapeutic goal can be achieved via AKG-induced downregulation of NEP and synergistic activity of AKG with thiorphan, i.e., a NEP specific inhibitor. Furthermore, this study has reported for the first time that exogenous AKG can influence the activity of histone methyltransferase, EZH2. However, this issue needs further investigation to elucidate the mechanisms of this phenomenon.

Acetylenic Synthetic Betulin Derivatives Inhibit Akt and Erk Kinases Activity, Trigger Apoptosis and Suppress Proliferation of Neuroblastoma and Rhabdomyosarcoma Cell Lines.

Neuroblastoma (NB) and rhabdomyosarcoma (RMS), the most common pediatric extracranial solid tumors, still represent an important clinical challenge since no effective treatment is available for metastatic and recurrent disease. Hence, there is an urgent need for the development of new chemotherapeutics to improve the outcome of patients. Betulin (Bet), a triterpenoid from the bark of birches, demonstrated interesting anti-cancer potential. The modification of natural phytochemicals with evidenced anti-tumor activity, including Bet, is one of the methods of receiving new compounds for potential implementation in oncological treatment. Here, we showed that two acetylenic synthetic Bet derivatives (ASBDs), EB5 and EB25/1, reduced the viability and proliferation of SK-N-AS and TE671 cells, as measured by MTT and BrdU tests, respectively. Moreover, ASBDs were also more cytotoxic than temozolomide (TMZ) and cisplatin (cis-diaminedichloroplatinum [II], CDDP) in vitro, and the combination of EB5 with CDDP enhanced anti-cancer effects. We also showed the slowdown of cell cycle progression at S/G2 phases mediated by EB5 using FACS flow cytometry. The decreased viability and proliferation of pediatric cancers cells after treatment with ASBDs was linked to the reduced activity of kinases Akt, Erk1/2 and p38 and the induction of apoptosis, as investigated using Western blotting and FACS. In addition, in silico analyses of the ADMET profile found EB5 to be a promising anti-cancer drug candidate that would benefit from further investigation.

Alpha Ketoglutarate Exerts In Vitro Anti-Osteosarcoma Effects through Inhibition of Cell Proliferation, Induction of Apoptosis via the JNK and Caspase 9-Dependent Mechanism, and Suppression of TGF-ß and VEGF Production and Metastatic Potential of Cells.

Osteosarcoma (OS) is the most common type of primary bone tumor. Currently, there are limited treatment options for metastatic OS. Alpha-ketoglutarate (AKG), i.e., a multifunctional intermediate of the Krebs cycle, is one of the central metabolic regulators of tumor fate and plays an important role in cancerogenesis and tumor progression. There is growing evidence suggesting that AKG may represent a novel adjuvant therapeutic opportunity in anti-cancer therapy. The present study was intended to check whether supplementation of Saos-2 and HOS osteosarcoma cell lines (harboring a TP53 mutation) with exogenous AKG exerted an anti-cancer effect. The results revealed that AKG inhibited the proliferation of both OS cell lines in a concentration-dependent manner. As evidenced by flow cytometry, AKG blocked cell cycle progression at the G1 stage in both cell lines, which was accompanied by a decreased level of cyclin D1 in HOS and increased expression of p21Waf1/Cip1 protein in Saos-2 cells (evaluated with the ELISA method). Moreover, AKG induced apoptotic cell death and caspase-3 activation in both OS cell lines (determined by cytometric analysis). Both the immunoblotting and cytometric analysis revealed that the AKG-induced apoptosis proceeded predominantly through activation of an intrinsic caspase 9-dependent apoptotic pathway and an increased Bax/Bcl-2 ratio. The apoptotic process in the AKG-treated cells was mediated via c-Jun N-terminal protein kinase (JNK) activation, as the specific inhibitor of this kinase partially rescued the cells from apoptotic death. In addition, the AKG treatment led to reduced activation of extracellular signal-regulated kinase (ERK1/2) and significant inhibition of cell migration and invasion in vitro concomitantly with decreased production of pro-metastatic transforming growth factor ß (TGF-ß) and pro-angiogenic vascular endothelial growth factor (VEGF) in both OS cell lines suggesting the anti-metastatic potential of this compound. In conclusion, we showed the anti-osteosarcoma potential of AKG and provided a rationale for a further study of the possible application of AKG in OS therapy.

Alpha ketoglutarate exerts a pro-osteogenic effect in osteoblast cell lines through activation of JNK and mTOR/S6K1/S6 signaling pathways.

Although numerous in vivo studies have suggested that alpha-ketoglutarate (AKG), i.e. the key intermediate in the Krebs cycle, may have an anabolic effect on bone tissue, the direct influence of AKG on osteoblasts and the underlying mechanism of its action have not been investigated so far. The aim of this study was to assess the impact of AKG (disodium salt dihydrate) on osteogenesis in vitro and identification of some signaling mechanisms involved in this activity. The human and mouse normal osteoblast cell lines hFOB 1.19 and MC3T3-E1 were used in this study. The results showed that AKG did not increase the proliferation of osteoblasts; however, it upregulated the expression of transcription factors RUNX2 and Osterix, the mRNA and protein levels of osteoblast differentiation markers (alkaline phosphatase, type I collagen, bone sialoprotein II, osteopontin, osteocalcin), and the mineralization levels in the hFOB 1.19 and MC3T3-E1 cell cultures. Moreover, AKG increased JNK, mTOR, S6K1, and S6 phosphorylation and decreased ERK1/2 phosphorylation in both osteoblast cell lines. The JNK inhibitor and rapamycin, but not the ERK inhibitor, abolished the AKG-promoted osteoblast differentiation. Using immunofluorescence staining, qRT-PCR, and Western blot analysis, we detected the presence of an AKG receptor GPR99 activated by alpha ketoglutaric acid in the tested osteoblast cell lines. However, AKG salt did not activate GPR99. Our findings suggest that AKG salt activates the JNK and mTOR/S6K1/S6 signaling pathways to promote differentiation of osteoblasts, independently of GPR99 activation. We can conclude that AKG salts might be promising candidates for bone anabolic drugs used for prevention or/and treatment of osteoporosis.

Betulin Promotes Differentiation of Human Osteoblasts In Vitro and Exerts an Osteoinductive Effect on the hFOB 1.19 Cell Line Through Activation of JNK, ERK1/2, and mTOR Kinases.

Although betulin (BET), a naturally occurring pentacyclic triterpene, has a variety of biological activities, its osteogenic potential has not been investigated so far. The aim of this study was to assess the effect of BET on differentiation of human osteoblasts (hFOB 1.19 and Saos-2 cells) in vitro in osteogenic (with ascorbic acid as an osteogenic supplement) and osteoinductive (without an additional osteogenic supplement) conditions. Osteoblast differentiation was evaluated based on the mRNA expression (RT-qPCR) of Runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), type I collagen-α1 (COL1A1), and osteopontin (OPN). Additionally, ALP activity and production of COL1A1 (western blot analysis) and OPN (ELISA) were evaluated. The level of mineralization (calcium accumulation) was determined with Alizarin red S staining. BET upregulated the mRNA level of RUNX2 and the expression of other osteoblast differentiation markers in both cell lines (except the influence of BET on ALP expression/activity in the Saos-2 cells). Moreover, it increased mineralization in both cell lines in the osteogenic conditions. BET also increased the mRNA level of osteoblast differentiation markers in both cell lines (except for ALP in the Saos-2 cells) in the osteoinductive conditions, which was accompanied with increased matrix mineralization. The osteoinductive activity of BET in the hFOB 1.19 cells was probably mediated via activation of MAPKs (JNK and ERK1/2) and mTOR, as the specific inhibitors of these kinases abolished the BET-induced osteoblast differentiation. Our results suggest that BET has the potential to enhance osteogenesis.

The molecular organization of prenylated flavonoid xanthohumol in DPPC multibilayers: X-ray diffraction and FTIR spectroscopic studies.

Xanthohumol (XN) is the major prenylated flavonoid found in hop resin. It has attracted considerable attention in recent years due to its wide spectrum of biological activities and the beneficial effect on human health. Since lipid membrane is first target for biologically active compounds, we decided to investigate the influence of XN on the dipalmitoylphosphatidylcholine (DPPC) multibilayers. Interactions of XN with DPPC were investigated as a function of temperature and its concentration by using X-ray diffraction and the ATR-FTIR spectroscopy techniques. The aim of understanding the mechanisms of molecular interactions between XN and DPPC was to indicate the localization of the XN with respect to the membrane and the type of interaction with phospholipids. The results revealed that XN changes the physical properties of the DPPC multibilayers in the form of dry film. A new complex formation between XN and DPPC is reported. The detailed analysis of refraction effect indicates the changes in electron density ratio between hydrophobic and hydrophilic zones of lipid at phase transition. This is in compliance with reported changes in FTIR spectra where at pretransition XN moves from interface region between polar heads to the neighborhood of phosphate groups.

Xanthohumol Impairs the PMA-Driven Invasive Behaviour of Lung Cancer Cell Line A549 and Exerts Anti-EMT Action.

Xanthohumol (XN), the main prenylated flavonoid from hop cones, has been recently reported to exert significant proapoptotic, anti-proliferative, and growth inhibitory effects against lung cancer in both in vitro and in vivo studies. However, its anti-metastatic potential towards this malignancy is still unrevealed. Previously, we indicated that the human lung adenocarcinoma A549 cell line was sensitive to XN treatment. Therefore, using the same tumour cell model, we have studied the influence of XN on the phorbol-12-myristate-13-acetate (PMA)-induced cell migration and invasion. The effects of XN on the expression/activity of pro-invasive MMP-9 and MMP-2 and the expression of MMP inhibitors, i.e., TIMP-1 and TIMP-2 (anti-angiogenic factors), were evaluated. Additionally, the influence of XN on the production of the key pro-angiogenic cytokine, i.e., VEGF, and the release of TGF-ß, which is both a pro-angiogenic cytokine and an epithelial-mesenchymal transition (EMT) stimulator, was studied. Furthermore, the influence of XN on the expression of EMT-associated proteins such as E-cadherin and α-E-catenin (epithelial markers), vimentin and N-cadherin (mesenchymal markers), and Snail-1 (transcriptional repressor of E-cadherin) was studied. To elucidate the molecular mechanism underpinning the XN-mediated inhibition of metastatic progression in PMA-activated cells, the phosphorylation levels of AKT, FAK, and ERK1/2 kinases, which are signalling molecules involved in EMT program activation, were assayed. The results showed that XN in non-cytotoxic concentrations impaired the PMA-driven migratory and invasive capacity of A549 cells by decreasing the level of expression of MMP-9 and concomitantly increasing the expression of the TIMP-1 protein, i.e., a specific blocker of pro-MMP-9 activation. Moreover, XN decreased the PMA-induced production of VEGF and TGF-ß. Furthermore, the XN-treatment counteracted the PMA-induced EMT of the A549 cells by the upregulation of E-cadherin and α-E-catenin and the downregulation of N-cadherin, vimentin, and Snail-1 expression. The proposed mechanism underlying the anti-invasive XN activity involved the inhibition of the ERK/MAPK pathway and suppression of FAK and PI3/AKT signalling. Our results suggesting migrastatic properties of XN against lung cancer cells require further verification in in vivo assays.

Prostate and breast cancer cells death induced by xanthohumol investigated with Fourier transform infrared spectroscopy.

Fourier Transform Infrared spectroscopy was applied to detect in vitro cell death induced in prostate (PC-3) and breast (T47D) cancer cell lines treated with xanthohumol (XN). After incubation of the cancer cells with XN, specific spectral shifts in the infrared spectra arising from selected cellular components were identified that reflected biochemical changes characteristic for apoptosis and necrosis. Detailed analysis of specific absorbance intensity ratios revealed the compositional changes in the secondary structure of proteins and membrane lipids. In this study, for the first time we examined the changes in these molecular components and linked them to deduce the involvement of molecular mechanisms in the XN-induced death of the selected cancer cells. We showed that XN concentration-dependent changes were attributed to phospholipid ester carbonyl groups, especially in the case of T47D cells, suggesting that XN acts as an inhibitor of cell proliferation. Additionally, we observed distinct changes in the region assigned to the absorption of DNA, which were correlated with a specific marker of cell death and dependent on the XN dose and the type of cancer cells. The microscopic observation and flow cytometry analysis revealed that the decrease in cancer cell viability was mainly related to the induction of necrotic cell death. Moreover, the T47D cells were slightly more sensitive to XN than the PC-3 cells. Considering the results obtained, it can be assumed that apoptosis and necrosis induced by XN may contribute to the anti-proliferative and cytotoxic properties of this flavonoid against cancer cell lines PC-3 and T47D.

Synthetic Betulin Derivatives Inhibit Growth of Glioma Cells In Vitro.

BACKGROUND/AIM: Glioma is the most malignant tumour of the human brain still lacking effective treatment modalities. Betulin, a pentacyclic triterpene abundantly found in the birch bark, has been shown to demonstrate interesting anti-cancer activity towards many cancer cells. We determined the effects of acetylenic synthetic betulin derivatives (ASBDs) as anti-tumour agents on glioma cells in vitro. MATERIALS AND METHODS: T98G and C6 glioma cell viability and proliferation were determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay and BrdU (bromo deoxyuridine) test, respectively. Cell-cycle progression and induction of apoptosis were investigated with flow cytometry. RESULTS: ASBDs significantly decreased glioma cell viability/survival and inhibited proliferation in a dose-dependent manner in vitro. Moreover, ASBDs were more cytotoxic than clinically used chemotherapeutics - temozolomide and cisplatin. CONCLUSION: ASBDs may be considered for further study as potent anti-tumour agents in glioma treatment.

Neutral endopeptidase (NEP) inhibitors - thiorphan, sialorphin, and its derivatives exert anti-proliferative activity towards colorectal cancer cells in vitro.

Neutral endopeptidase (NEP) is an enzyme implicated in development of different tumors, e.g. colorectal cancer (CRC). In this study, the anti-cancer effects of NEP inhibitors, thiorphan (synthetic compound) and sialorphin (naturally occurring pentapeptide) on CRC cells were investigated. Moreover, we synthesized some derivatives of sialorphin (alanine scan analogues: AHNPR, QANPR, QHAPR, QHNAR; N-acetylated sialorphin; C-amidated sialorphin, and C-amidated alanine scan analogues) to examine the biological activity of these inhibitors on CRC cells. The cytotoxic activity of the NEP inhibitors against CRC cell lines (SW620 and LS180) and normal human fibroblasts (HSF) was evaluated. Additionally, the influence of NEP inhibitors on proliferation, cell cycle progression, induction of apoptosis, and the level of phosphorylation of MAP kinases and mTORC1 signaling pathway proteins in CRC cells were examined. The NEP inhibitors were non-cytotoxic to HSF cells; however, most of them slightly decreased the viability and inhibited proliferation of CRC cells. The N-acetylation or C-amidation of sialorphin or its alanine scan analogues resulted in decreased or abolished anti-proliferative activity of the NEP inhibitors towards the CRC cells. Additionally, thiorphan and sialorphin enhanced the anti-proliferative activity of other CRC-cell growth inhibitors (atrial natriuretic peptide-ANP and melphalan-MEL). The mechanisms involved in the anti-proliferative effects of the tested inhibitors were mediated via NEP and associated with induction of cell cycle arrest in the G0/G1 phase, increased activity of ERK1/2, and a reduced level of phosphorylation of mTOR (Ser2448), 4E-BP1, and p70S6K. However, the NEP inhibitors did not induce apoptosis in the CRC cells. These results have indicated that thiorphan and sialorphin or its derivatives AHNPR, QANPR, QHAPR, and QHNAR have the potential to be used as agents in treatment of patients with CRC.

Xanthohumol exhibits anti-myeloma activity in vitro through inhibition of cell proliferation, induction of apoptosis via the ERK and JNK-dependent mechanism, and suppression of sIL-6R and VEGF production.

BACKGROUND: Xanthohumol (XN, a hop-derived prenylflavonoid) was found to exert anticancer effects on various cancer types. However, the mechanisms by which XN affects the survival of multiple myeloma cells (MM) are little known. Therefore, our study was undertaken to address this issue. METHODS: Anti-proliferative activity of XN towards two phenotypically distinct MM cell lines U266 and RPMI8226 was evaluated with the MTT and BrdU assays. Cytotoxicity was determined with the LDH method, whereas apoptosis was assessed by flow cytometry and fluorescence staining. The expression of cell cycle- and apoptosis-related proteins and the activation status of signaling pathways were estimated by immunoblotting and ELISA assays. RESULTS: XN reduced the viability of RPMI8226 cells more potently than in U266 cells. It blocked cell cycle progression through downregulation of cyclin D1 and increased p21 expression. The marked apoptosis induction in the XN-treated RPMI8226 cells was related to initiation of mitochondrial and extrinsic pathways, as indicated by the altered p53, Bax, and Bcl-2 protein expression, cleavage of procaspase 8 and 9, and elevated caspase-3 activity. The apoptotic process was probably mediated via ROS overproduction and MAPK (ERK and JNK) activation as N-acetylcysteine, or specific inhibitors of these kinases prevented the XN-induced caspase-3 activity and, hence, apoptosis. Moreover, XN decreased sIL-6R and VEGF production in the studied cells. CONCLUSIONS: ERK and JNK signaling pathways are involved in XN-induced cytotoxicity against MM cells. GENERAL SIGNIFICANCE: The advanced understanding of the molecular mechanisms of XN action can be useful in developing therapeutic strategies to treat multiple myeloma.

Anticancer effect of the water extract of a commercial Spirulina (Arthrospira platensis) product on the human lung cancer A549 cell line.

Spirulina is a well-described and popular dietary supplement derived from Arthrospira algae. In the present study, the anticancer potential of a water extract of a commercial Spirulina product (SE) against the human non-small-cell lung carcinoma A549 cell line was evaluated. After qualitative analysis, we investigated the effect of SE on cell viability, proliferation, and morphology. Furthermore, the influence of SE on regulation of the cell cycle, induction of apoptosis in lung cancer cells, and expression of cell cycle/apoptosis-related proteins was evaluated. Additionally, we examined the cytotoxic effect of SE on normal human skin fibroblasts (HSF). Our studies revealed that SE significantly reduced cancer cell viability and proliferation, which was accompanied by cell cycle inhibition in the G1 phase, induction of apoptosis, and prominent morphological changes. Moreover, we detected no cytotoxic effect of the tested Spirulina extract on normal skin fibroblasts. Our molecular studies demonstrated that SE reduced the phosphorylation of Akt and Rb proteins, reduced the expression of cyclin D1 and CDK4, and increased the Bax to Bcl-2 ratio in the A549 cells. In conclusion, the results obtained provide evidence of the anti-cancer activity of the commercial Spirulina product against lung cancer cells and strongly support the knowledge of the chemopreventive properties of Spirulina.